The present invention relates to novel thermoprecipitating polymers containing enzyme specific ligands. The present invention also relates to a process for the preparation of such enzyme specific ligands and to monomers embodying such ligands. More particularly, the present invention also relates to a process for the separation of enzymes using such thermoprecipitating polymers. The invention also relates to a process for the separation of lysozyme and lectins from a mixture of proteins and lectins such as wheat germ agglutinin and tomato agglutinin.
The separation and recovery of biomolecules like enzymes and glycoproteins are critical cost determining steps in most of the down stream processes in biotechnology industries. Conventionally, separation of lysozyme from the crude sources has been done by salt precipitation (Hasegawa, Mineo, Yoshida, Kazuya, Miyauchi, Sakae, Terazono, Masami: U.S. Pat. No. 4,504,583, (1985)) or by ion exchange techniques (Hasegawa, Mineo, Ozaki, Kitao: U.S. Pat. No. 4,705,755 (1987); Durance, Timothy, L-Chan, Eunice, Nakai, Shuryo; U.S. Pat. No. 4,966,851 (1990); Hasegawa, Mineo; U.S. Pat. No. 4,518,695 (1985); Takechi, Kaz, Takahashi, Tsuyos, Inaba, Toyoaki, Hasegawa, Eiichi: U.S. Pat. No. 4,104,125 (1978).
Various techniques based on affinity interactions between enzymexe2x80x94inhibitor, enzymexe2x80x94substrate, enzymexe2x80x94transition state analog, enzymexe2x80x94cofactor and the like have been developed as better alternatives to the above-mentioned conventional systems for the selective recovery of enzymes. Most of the affinity based separations involve polymers to which the affinity ligand or cofactors or dyes are chemically linked. The complex formed between enzyme and the polymeric ligand is subsequently processed to isolate the enzyme.
These alternative systems primarily involve affinity chromatography or affinity ultrafiltration. Although these techniques provide high selectivity, they are beset with several practical difficulties which are summarised below.
Bozzano A. G., Glatz C. E., J. Memb. Sci 55, 181-198 (1991); Ehsani N., Parkkinen S., Nystrom M. J. Memb Sci 123, 105-119, (1997) disclose that affinity ultrafiltration is suitable for the separation of enzyme only in cases where the difference between the molecular weights of the desired enzyme and other biomolecules is appreciably large. Also, at high pressures, the denaturation of the enzyme and fouling of the membrane leads to poor product quality (Ehsani N., Parkkinen S., Nystrom M. J. Memb Sci 123, 105-119, (1997)).
Affinity chromatography (Hirano S., Kaneko H., Kitagawa M., Agric. Biol. Chem, 55, 1683-1684 (1991); Safrik L., Safarikova M., J. Biochem Biophys. Methods, 27, 327-330 (1993); Reid, Lorne S.: U.S. Pat. No. 4,552,845 (1985)) is found suitable only for small capacity columns. Scale up of the columns retards the flow rate and leads to clogging of the columns, thereby resulting in increased cost of the process. Another disadvantage of this process is that it is non-continuous since periodic flushing is required at specified time intervals to remove any undesirable non-specifically adsorbed biomolecules in the column (Chern C. S., Lee C. K., Chen C. Y., Yeh M. J., Colloids and Surfaces B. Biointerfaces, 637-49, (1996)).
Affinity precipitation eliminates many of the above-mentioned problems and offers certain advantages such as ease of scale up, amenability to continuous operation and recycling of the affinity ligand (Chern C. S., Lee C. K., Chen C. Y., Yeh M. J., Colloids and Surfaces B. Biointerfaces. 637-49, (1996)). Affinity precipitation involves the formation of a complex between the enzyme and stimuli sensitive affinity polymer in a homogeneous solution. This complex is precipitated by a change in the pH, temperature or ionic strength. This complex is dissociated and the polymer is separated by varying either one of the above-mentioned stimuli and the enzyme is then isolated (Gupta M. N., Kaul R., Guogiang D., and Mattiasson B., J. Mol. Recog. 9, 356-359, (1996)). Thus the recovery of the enzyme by this process is much simpler.
It is well known that ligands containing N-acetyl groups such as N-acetylglucosamine, N-acetylmuramic acid, chitin, chitosan, and the like exhibit affinity for various enzymes and lectins (Tyagi R., Kumar A., Sardar M., Kumar S., Gupta M. N., Isol. Purif. 2, 217-226 (1996); Katz, Friedrich D., Fishman, Louis, Levy; U.S. Pat. No.: 3,940,317 (1976)).
All the above ligands are derived from glucose. Since glucose is a carbons source for many microbes, such ligands are susceptible to microbial attack and hydrolytic degradation resulting in poor stability of these ligands (Hirano S., Kaneko H., Kitagawa M., Agric. Biol. Chem. 55, 1683-1684 (1991)). Also chitosan is insoluble in alkaline media while crosslinked chitosan and chitin are insoluble in both alkaline and acidic media (Ruckstein, Eli; Zeng, Xianfang; Biotechnology and Bioengineering, 56, 610-617 (1997)). This limits their use. Moreover, chitin and chitosan can undergo transglycosylation and mutarotation, which drastically reduces their efficiency in any affinity based recovery of enzyme (Davies, R. C., Neuberger, A., Wilson, B. M., Biochem. Biophys Acta. 178, 294-305, (1969); Neuberger, A., Wilson, B. M. Biochem. Biophys. Acta, 147, 473-486, (1967)). Thus, it is desirable to replace glucose by stable synthetic ligands during the synthesis of thermoprecipitating affinity polymers for enzyme separations.
It is an object of the invention to provide thermoprecipitating polymers and processes for the preparation thereof, said thermoprecipitating polymers containing ligands having N-acetyl groups.
It is another object of the invention to provide stable synthetic ligands for thermoprecipitating polymers for enzyme separation.
It is another object of the invention, to provide acrylic monomers and process for the preparation thereof which are useful in the synthesis of thermoprecipitating polymers containing the above ligands having N-acetyl groups.
It is a further object of the invention to provide an economical process for the separation of lysozyme using the thermoprecipitating polymers of the invention.